Osmotic uptake of water is the driving force of plant cell expansion. As water enters the cell, the protoplast expands but is restrained by the cell wall. Moreover, a rigid complex of cellulose microfibril polymers embedded in a glue-like matrix of pectins, hemicelluloses and proteins forms part of this wall in mature cells. It has long been thought that some xe2x80x9cwall looseningxe2x80x9d factor must be present which alters immature cell wall mechanical properties and allows it to undergo a process of elongation. McQueen-Mason et al., Plant Cell, Vol. 4, pp. 1425-1433 (1992) studied plant cell enlargement regulation by employing a reconstitution approach. The authors found that a crude protein extract from the cell walls of growing cucumber seedlings possess the ability to induce the extension of isolated cell walls. Sequential HPLC fractionation of the active wall extract revealed two proteins with molecular masses of 29 and 30 kD associated with the activity. Each protein, by itself, could induce wall extension without detectable hydrolytic breakdown of the wall and appeared to mediate xe2x80x9cacid growthxe2x80x9d responses of isolated walls and may catalyze plant cell wall extension by a novel biochemical mechanism.
Shcherban et al., Proc. Nat. Acad. Sci., USA, Vol. 92, pp. 9245-9249 (1995) isolated cDNA""s encoding these two cucumber proteins and compared them to anonymous expressed sequence tags from various sources. Rice and Arabidopsis expansin cDNA were identified from these collections and showed at least four different expansin cDNA""s in rice and six different expansin cDNA""s in Arabidopsis. The authors concluded that expansin are highly conserved in size and sequence (60-87% amino acid identity and 75-95% similarity between any pairwise comparison) and that the multigene family formed before the evolutionary divergence between monocotyledons and dicotyledons. Shcherban et al. states that the high conservation of this mutligene family indicates that the mechanism by which expansin promotes cell wall extension tolerates little variation in protein structure.
Wang et al., Biotech. Lett., Vol. 16, No. 9, pp. 955-958 (1994) discovered two proteins in a Chinese medicinal cucumber, Trichosanthes kirilowii, which appear to be similar to the S1 and S2 proteins which demonstrate cell wall extension properties. Similar proteins were also found in growing tomato leaves (Keller et al., The Plant Journal, Vol. 8, No. 6, pp. 795-802 (1995)) and in oat coleoptile walls (Li et al., Planta, Vol. 191, pp. 349-356 (1993)).
Cosgrove et al., J. Exp. Botany, Vol. 45, Special Issue, pp. 1711-1719 (1994) suggested that cooperative interactions between the expansin proteins and pectinases and cellulases may occur, wherein the enzymes modify the matrix so that other wall extension mechanisms may be more effective. Fry, Current Biology, Vol. 4, No. 9 (1994) suggest that, in loosening cell walls, expansin seems unlikely to break cellulose-cellulose bonds as microfibrils remain intact during growth. Thus, the authors discount the observed breakage of hydrogen bonds in filter paper as a side issue and suggest that expansin may lengthen inter-microfibrillar tethers by causing hemicellulose chains to detach from cellulose microfibrils to allow extension.
Despite the pioneering work previously done in the area of cell wall extension and its causes, work related to the usefulness and operability of expansins is still in its infancy. Moreover, the sources of expansin up to now have been exclusively from plant origins, for which expression systems may not be optimal for large scale production. Accordingly, it would be valuable to have a ready source of expansin-like material which is capable of being produce in large quantities from organisms which are established high output producers of biological materials, such as fungi, bacteria or other well characterized microorganisms.
It is an object of the present invention to provide for a swollenin protein which is derived from a microbial non-plant source.
It is another object of the present invention to provide for a swollenin protein which is expressible in a well-characterized microorganism, for example a fungus or bacteria, so as to facilitate its production in large quantities.
It is yet another object of the present invention to provide a DNA sequence corresponding to a microbial swollenin which can be used in industrial production of swollenin protein.
It is yet another object of the present invention to provide for novel and useful methods of altering cellulosic substrates, such as pulp and paper, cellulose based textile fibers, animal feed and corn wet milling or dry milling polysaccharide waste products or other cellulosic biomass.
According to the present invention, a partially or wholly isolated swollenin protein derived from a fungus or bacteria is provided. Preferably, the swollenin is derived from a filamentous fungus, more preferably, from a filamentous fungus such as Trichoderma spp., Humicola spp., Neurospora spp., Aspergillus spp., Fusarium spp., Penicillium spp., or Gliocladium spp. and most preferably, from Trichoderma spp. In a particularly preferred embodiment of the present invention, the swollenin comprises a sequence according to SEQ. ID NO:2, has at least 70% sequence identity with the sequence provided in SEQ. ID NO:2 or comprises a derivative of the sequence according to SEQ. ID NO:2, wherein the swollenin further has the ability to weaken filter paper and/or swell cotton fibers.
In another embodiment of the present invention, a DNA is provided encoding a swollenin protein from a fungus or bacteria. Preferably, the DNA is derived from a filamentous fungus such as Trichoderma spp., Humicola spp., Neurospora spp., Aspergillus spp., Fusarium spp., Penicillium spp., or Gliocladium spp. Also preferably, the DNA comprises the sequence according to SEQ. ID. NO:1. Alternately, the DNA has at least 70% sequence identity with the sequence according to SEQ. ID NO:1 or comprises a derivative of the sequence according to SEQ. ID NO:1, wherein said DNA encodes a swollenin protein which has the ability to weaken filter paper and/or swell cotton fibers. In a preferred embodiment of the invention, the DNA hybridizes with a DNA having all or part of the sequence provided in SEQ ID NO:1.
In another embodiment of the invention, a DNA is provided which encodes a microbial, e.g., bacterial or fungal, swollenin, and the DNA hybridizes with a DNA probe encoding a peptide having an amino acid sequence comprising SEQ. ID NO:14, SEQ. ID NO:15, SEQ. ID NO:16, SEQ. ID NO:17 or SEQ. ID NO:18. Vectors comprising such DNA, host cells having been transformed with such vectors and fermentation broths produced by such transformed host cells are also within the scope of the present invention.
In yet another embodiment of the present invention, a method of producing swollenin protein is provided comprising the steps of (a) obtaining a host cell which has been transformed with a vector comprising DNA encoding a swollenin protein, the DNA being isolated from a fungus or bacteria; (b) culturing the host cell under conditions suitable for the expression and, optionally, secretion, of the swollenin protein; and (c) recovering the fermentation broth containing said swollenin protein.
Since fungi and bacteria do not generally have a cellulosic cell wall and in any event are not known to increase in size by the same mechanism as higher plants, Applicants discovery that these microorganisms produce proteins having expansin-like properties is not suggested by previous work related to plant expansins. Thus, the finding that the cellulolytic fungus Trichoderma spp. produces an expansin-like protein is unexpected. However, it is apparent that the microbial class of proteins differs from those heretofore discovered in plants. For example, the presence of a region on the microbial swollenin protein described herein corresponding to the cellulose binding domain of fungal cellulolytic enzymes suggests that this protein is secreted to act in concert with the naturally secreted cellulases and hemicellulases in order to facilitate hydrolysis of cellulosic biomass in the environment. Consistent with this suggestion, the Trichoderma reesei swollenin gene was found to be expressed when the fungus was grown on cellulose as a sole carbon source, but not when the carbon source for growth was glucose. This pattern of regulation of gene expression is similar to that observed for many of the Trichoderma cellulose and hemicellulose genes. These unexpected findings lead to the conclusion that cellulose or hemicellulose degrading micro-organisms, including bacteria, yeast and fungi, would also produce- such swollenin proteins.
Accordingly, it is an advantage of the present invention that the swollenins provided herein may have utility in many applications for which cellulase is currently used, for example, cleaning textiles (laundry detergents and pre-wash compositions), modifying textiles (depilling, color restoration, anti-greying), stonewashing denim, biomass conversion to glucose, and improvement of the nutritive value of animal feeds. Similarly, it is contemplated that an advantage of the present invention is that swollenins may have a synergistic or additive effect in combination with other enzymes, particularly cellulases such as endoglucanases. In other cases, it is possible that swollenins would have a deleterious effect in an application; for example, they may cause excessive fabric strength loss when present as a side activity in an endoglucanase produced by fermentation of a microorganism and used for fabric cleaning or modification. In such a case, removal of the swollenin from a cellulase product may be beneficial and may be accomplished by biochemically removing the product from the resultant cellulase mixture, through genetic engineering to prevent its expression or to inactivate the gene or by adding a chemical inhibitor to the composition comprising the swollenin.